Molecular aging is marked by the accumulation of covalently modified polypeptides. Survival during aging therefore depends in part on limiting the accumulation of proteins damaged by spontaneous chemical reactions to maintain homeostasis. Examples of such damages include the non-enzymatic modification by the oxidation of various residues, the glycation of lysine residues, and the deamidation and isomerization of asparagine and aspartyl residues into L-isoaspartyl residues. To date, enzymatic recognition of these damages followed by subsequent repair has been the most well-characterized mechanism for regulating age-related damages which include the action of methionine sulfoxide reductases, ketoamine repair enzymes, and the protein-L-isoaspartyl (D-aspartyl) O-methyltransferase that was discovered in our laboratory (PCMT1).

Protein homeostasis is also maintained by the equilibrium of protein synthesis and protein turnover to maintain a functional proteome; the majority of that protein turnover is regulated by the ubiquitin-proteosome system (UPS). Within the UPS, E3 Ubiquitin (Ub) ligases facilitate the degradation of proteins by recognizing specific targets. This recognition is mediated by substrate-specific sequence motifs known as degrons. Degron binding can be further regulated by enzyme-catalyzed post-translational modifications (PTMs) that either promote or inhibit substrate-E3 Ub ligase interactions. However, evidence is sparse for a general class of E3 Ub ligases that recognizes degrons defined by non-enzymatically-derived PTMs.

With the long-term interest of our laboratory in a major type of spontaneous protein damage that accumulates with age – the formation of kinked polypeptide chains with L-isoaspartyl residues as described above, we are now interested in both repair and potential degradation pathways. Mitigating this type of protein damage is necessary for maintaining proteome stability and prolonging organismal survival. As described above, the damaging L-isoaspartate modification has often been observed to have a negative effect on protein function, but can be repaired in many proteins by the protein-L-isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1). Significantly, previous studies in our laboratory have demonstrated that L-isoaspartate levels unexpectedly plateaued in Pcmt1 knockout mice that are transgenic with a mouse Pcmt1 cDNA under a neuron-specific promoter, while levels of L-isoaspartate containing peptides in the urine increased. We then hypothesized that a secondary maintenance pathway exists which recognizes L-isoaspartate-damaged proteins and initiates their degradation but the nature of this pathway remained elusive for many years.

Recently, we have been intrigued to note two distinct human genes that encode proteins with unusual similarity to the repair methyltransferase PCMT1. A gene on chromosome 8 encodes PCMTD1 (Protein-L-isoaspartate O-methyltransferase domain-containing protein 1) and a gene on chromosome 20 encodes PCMTD2. Significantly, in addition to the AdoMet binding motifs, sequence similarity is seen in two regions known to be involved in binding the L-isoaspartyl substrate of PCMT1. Sequence comparisons of the C-terminal domain has demonstrated similarity to motifs in ubiquitin E3 ligases. These results led us to hypothesize that PCMTD1 and PCMTD2 may function to recognize damaged proteins using their N-terminal PIMT methyltransferase-like domain and then bring these proteins to ubiquitin-coupling proteins via the C-terminal domain for proteasomal degradation. Such a mechanism would provide mammalian cells a backup system for limiting the accumulation of damaged proteins in aging.

Of the E3 Ub ligases, cullin-RING ligases (CRLs) represent the largest subclass of such enzymes. CRLs feature a conserved catalytic core formed by a cullin protein and a RING protein. Acting as scaffolds, cullins link the RING protein at its C-terminus with a diverse pool of substrate receptors at its N-terminus. Receptors are able to bind specific substrates while interacting with cullins through specific motifs. For CUL5 CRLs, these substrate receptors consist of suppressor of cytokine signaling (SOCS)-box proteins that utilize an Elongins B/C heterodimer (ELOBC) to associate with the N-terminus of CUL5 via the BC-box and Cul-box motifs, respectively. Ubiquitylation of targets is driven by the spatial and temporal coordination of receptor-bound substrates with ubiquitylation machinery recruited by RING proteins.

In collaboration with Professors Jose Rodriguez and Joseph Loo, we have now shown the atomic structure of the human PCMTD1-Elongin B-Elongin C-Cullin 5-Rbx2 E3 ligase complex (A336). Importantly, we showed that this PCMTD1 CRL complex specifically binds L-isoaspartyl residues when bound to AdoMet. PCMTD1 may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein. This work provides evidence for a growing class of E3 ubiquitin ligases that recognize spontaneous covalent modifications as potential substrates for ubiquitylation and subsequent proteasomal degradation.

Publication: Pang, E. Z., Zhao, B., Flowers, C., Oroudjeva, E., Winter, J. B., Pandey, V., Sawaya, M. R., Wohlschlegel, J., Loo, J. A., Rodriguez, J. A., and Clarke, S. G. (2025) “Structural basis for L-isoaspartyl-containing protein recognition by the human PCMTD1 cullin-RING E3 ubiquitin ligase” J. Biol. Chem. 301, article 110735, pp.1-17. (DOI: 10.1016/j.jbc.2025.110735). PMID: 40975169. PMCID: PMC12590147. bioRxiv (Preprint) 2025 May 21:2025.05.21.654933. doi: 10.1101/2025.05.21.654933. PMID: 40475564. PMCID: PMC12139721.

We are now pursuing the function of the PCMTD1 and PCMTD2 proteins in recognizing age-damaged proteins. We are now poised to expand on our previous identification of a complex of PCMTD1 with elongins B and C, cul5, and Rbx2. In a collaboration with Professor Joseph Loo’s mass spectrometry laboratory at UCLA, we will use his newly developed techniques for top-down mass spectrometry to detect the binding of proteins and peptides containing damaged aspartyl residues to the complex. These studies will then make possible direct studies of the ubiquinylation of the damaged species and their degradation.